In the brain, the chemical acetylcholine (ACh) exerts powerful modulatory control over arousal, motor and cognitive circuits, and has been found to be deficient in Alzheimer's Disease (AD). The current drugs available to positively impact cognitive deficits in Alzheimer's Disease (AD) and other dementias are the cholinesterase inhibitors. These prevent the breakdown of the neurotransmitter acetylcholine (ACh), and thus augment Ach function. Due to the limited utility of the cholinesterase inhibitors, alternative therapies to augment ACh deficits are critical in our aging population. Another vital protein, the hemicholinium-3 sensitive choline transporter (CHT) is believed to be responsible for the efficient uptake of choline by neurons to allow for ACh synthesis. We have developed an assay system for high throughput screening to identify compounds with high selectivity for CHT. It is anticipated that these compounds may lead to future cholinergic therapies in AD, and multiple other CNS diseases regulated by cholinergic signaling. Our research aims are to implement a high throughput screen for discovery of novel enhancers of high-affinity choline transporter uptake as mediated by CHT using voltage-sensitive fluorescent measurements;to perform secondary assays aimed at identifying novel modulators that act selectively at the choline transporter (CHT);and to use database mining and chemical synthesis to optimize HTS hits for use in biological assays. Through the development of high-throughput screening (HTS) methodologies that allow for the screening of large chemical libraries, we seek to identify CHT activators and allosteric modulators. These compounds may be able to enhance choline uptake and increase the levels of ACh produced in the neuron. Such reagents would allow greater ACh release from viable neurons not lost in the disease process and could offer advantages over cholinesterase therapy in targeting precursors rather than ACh itself. PUBLIC HEALTH RELEVANCE: Cognitive deficits associated with Alzheimer's Disease (AD) and other dementias are primarily caused by loss of the neurotransmitter acetylcholine (ACh) in the brain and treatments are currently limited to cholinesterase inhibitors. Additional and improved therapies to augment ACh deficits are critical in our aging population and may be accessed by targeting another vital protein, the hemicholinium-3 sensitive choline transporter (CHT). CHT is responsible for the efficient uptake of choline by neurons and we have developed an assay for high throughput screening to identify compounds with high selectivity for CHT for future cholinergic therapies in AD, and other diseases regulated by cholinergic signaling.